Ligand binding and protein dynamics in neuroglobin

Abstract

Neuroglobin (Ngb) is a recently discovered protein in vertebrate brain tissue that belongs to the globin family of proteins. It has been implicated in the neuronal response to hypoxia or ischemia, although its physiological role has been hitherto unknown. Ngb is hexacoordinate in the ferrous deoxy form under physiological conditions. To bind exogenous ligands like O2 and CO, the His E7 endogenous ligand is displaced from the sixth coordination. By using infrared spectroscopy and nanosecond time-resolved visible spectroscopy, we have investigated the ligand-binding reaction over a wide temperature range (3-353 K). Multiple, intrinsically heterogeneous distal heme pocket conformations exist in NgbCO. Photolysis at cryogenic temperatures creates a five-coordinate deoxy species with very low geminate-rebinding barriers. The photodissociated CO is observed to migrate within the distal heme pocket even at 20 K. Flash photolysis near physiological temperature (275-353 K) exhibits four sequential kinetic features: (i) geminate rebinding (t < 1 s); (ii) extremely fast bimolecular exogenous ligand binding (10 s < t < 1 ms) with a nontrivial temperature dependence; (iii) endogenous ligand binding (100 s < t < 10 ms), which can be studied by using flash photolysis on deoxy Ngb; and (iv) displacement of the endogenous by the exogenous ligand (10 ms < t < 10 ks). All four processes are markedly nonexponential, suggesting that Ngb fluctuates among different conformations on surprisingly long time scales.G lobins are proteins that bind dioxygen and other small ligands at the central iron of a heme prosthetic group embedded in a highly conserved ␣-helical ''globin'' fold. Hemoglobin (Hb) and myoglobin (Mb) are the most prominent members of this protein family (1). Interrelations among structure, dynamics, and function in globins have been investigated in great detail, most likely more thoroughly than for any other protein family. Mb especially has, for a long time, served as a paradigm in the biological physics of proteins (2, 3).Recently, two new members have joined the globin family, cytoglobin (4) and neuroglobin (Ngb) (5). The latter consists of a single chain of 151 aa. Ngb, which occurs in neurons, has less than 25% sequence identity with Mb or Hb but nevertheless displays all key determinants of the globin fold (6). It has moderate oxygen affinity and seems most closely related to the globin found in the glial cells of the annelid Aphrodite (7). The discovery of a six-coordinate heme in the deoxy form of Ngb (5), with the His E7 side chain occupying the sixth coordination, came somewhat as a surprise because it was believed for a long time that a pentacoordinate heme iron, with a vacant ligandbinding site, was a common characteristic of globins. In recent years, however, hexacoordinate globins also have been isolated from bacteria, unicellular eukaryotes, and plants [nonsymbiotic Hbs, nsHbs (8,9), and truncated Hbs, trHbs (10)]. In these proteins, the exogenous ligand has to compete with an intramolecular side chain for ...

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“…The physiological role of these hexacoordinated Hbs is not well understood. Several roles have been suggested for them: oxygen scavengers under hypoxic conditions [56,57], terminal oxidases [58], oxygen sensors [59,60], and that they are involved in nitrogen monoxide metabolism [61].…”

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“…While hxHbs can bind oxygen and other exogenous ligands, kinetic and equilibrium analysis of the reactions is much more complex than the bimolecular reactions observed with pentacoordinate Hbs due to competition from intramolecular coordination of the binding site. While many functions have been proposed for hxHbs from different organisms, and they have the biochemical capabilities to perform many reactions (4), no clear role has yet been supported to the exclusion of others (6,(8)(9)(10)(11)(12).…”

Influence of Distal Residue B10 on CO Dynamics in Myoglobin and Neuroglobin

“…In comparison with Mb, much less is known about the structural details of exogenous ligand binding in Ngb [41,42]. It involves an additional step, the dissociation of the distal HisE7 side chain to make the sixth coordination site available for an external ligand.…”